The present invention relates to nucleic acids which encode glycosyltransferase and are useful in producing cells and organs from one species which may be used for transplantation into a recipient of another species. Specifically the invention concerns production of nucleic acids which, when present in cells of a transplanted organ result in reduced levels of antibody recognition of the transplanted organ.
The transplantation of organs is now possible due to major advances in surgical and other techniques. However, availability of suitable human organs for transplantation is a significant problem. Demand outstrips supply. This has caused researchers to investigate the possibility of using non-human organs for transplantation.
Xenotransplantation is the transplantation of organs from one species to a recipient of a different species. Rejection of the transplant in such cases is a particular problem, especially where the donor species is more distantly related, such as donor organs from pigs and sheep to human recipients. Vascular organs present a special difficulty because of hyperacute rejection (HAR).
HAR occurs when the complement cascade in the recipient is initiated by binding of antibodies to donor endothelial cells.
Previous attempts to prevent HAR have focused on two strategies: modifying the immune system of the host by inhibition of systemic complement formation (1,2) and antibody depletion (3,4). Both strategies have been shown to temporarily prolong xenograft survival. However, these methodologies are therapeutically unattractive in that they are clinically impractical and would require chronic immunosuppressive treatments. Therefore, recent efforts to inhibit HAR have focused on genetically modifying the donor xenograft. One such strategy has been to achieve high-level expression of species-restricted human complement inhibitory proteins in vascularized pig organs via transgenic engineering (5-7). This strategy has proven to be useful in that it has resulted in the prolonged survival of porcine tissues following antibody and serum challenge (5,6). Although increased survival of the transgenic tissues was observed, long-term graft survival was not achieved (6). As observed in these experiments and also with systemic complement depletion, organ failure appears to be related to an acute antibody-dependent vasculitis (1,5).
In addition to strategies aimed at blocking complement activation on the vascular endothelial cell surface of the xenograft, recent attention has focused on identification of the predominant xenogeneic epitope recognised by high-titre human natural antibodies. It is now accepted that the terminal galactosyl residue, Gal-xcex1(1,3)-Gal, is the dominant xenogeneic epitope (8-15). This epitope is absent in Old World primates and humans because the xcex1(1,3)-galactosyltransferase (gal-transferase or GT) is non-functional in these species. DNA sequence comparison of the human gene to xcex1(1,3)-galactosyltransferase genes from the mouse (16,17), ox (18), and pig (12) has revealed that the human gene contained two frameshift mutations, resulting in a non-functional pseudogene (20,21). Consequently, humans and Old World primates have pre-existing high-titre antibodies directed at this Gal-xcex1(1,3)-Gal moiety as the dominant xenogeneic epitope.
It appears that different glycosyltransferases can compete for the same substrate. Hence xcex1(1,2)-fucosyltransferase or H transferase (HT) (22) could be an appropriate enzyme to decrease the expression of Gal-xcex1(1,3)-Gal, as both the xcex1(1,2)-fucosyltransferase and the xcex1(1,3)-galactosyltransferase use N-acetyl lactosamine as an acceptor substrate, transferring fucose or galactose to generate fucosylated N-acetyl lactosamine (H substance) or Gal-xcex1(1,3)-Gal, respectively. Furthermore, the xcex1(1,3)-galactosyltransferase of most animals cannot use the fucosylated N-acetyl lactosamine as an acceptor to transfer the terminal galactose, but will only transfer to N-acetyl lactosamine residues (23). We have previously reported that the simultaneous expression of two glycosyltransferases, xcex1(1,2)-fucosyltransferase (H transferase) and xcex1(1,3)-galactosyltransferase, does not lead to equal synthesis of each monosaccharide, but the activity of the xcex1(1,2)-fucosyltransferase is given preference over that of the xcex1(1,3)-galactosyltransferase, so that the expression of Gal-xcex1(1,3)-Gal is almost entirely suppressed (24).
The xcex1(1,3)-galactosyltransferase (Gal transferase) can galactosylate two types of precursor chains: Type 1: Galxcex2(1,3)GlcNAc and Type 2: Galxcex2(1,4)GlcNAc.
Furthermore, both of these precursors can be transformed into H substance or fucosylated xcex2-D-Gal by two xcex1(1,2)-fucosyltransferases (25,26). These two fucosyltransferases are H-transferase or FUT1 (22) and secretor (Se) transferase or FUT2 (27). While both enzymes can use both types of precursors, FUT1 HT preferentially utilises Type 2 precursor chains, and FUT2 preferentially utilises Type 1 (28).
In work leading up to the present invention the inventors set out to create a nucleic acid which would be useful in reducing unwanted carbohydrate epitopes on the surface of cells. The nucleic acid could be used in production of an organ which would cause reduced levels of rejection when transplanted into another species. The inventors surprisingly found that a glycosyltransferase derived from porcine origin was useful in decreasing unwanted carbohydrate epitopes in cells. The enzyme encoded by the nucleic acid is able to compete effectively with glycosyltransferases which produce unwanted carbohydrate epitopes. In this particular work the inventors cloned a secretor transferase (Se) gene from pig origin, and demonstrated that this is expressed in cells and results in reduced levels of unwanted epitopes on those cells. The secretor transferase is referred to herein as xe2x80x9cpig secretorxe2x80x9d.
In a first aspect the invention provides a nucleic acid encoding a first glycosyltransferase which is able to compete with a second glycosyltransferase for a substrate when said nucleic acid is expressed in a cell which produces said second glycosyltransferase, resulting in reduced levels of a product from said second glycosyltransferase.
The nucleic acid may be DNA or RNA, single or double stranded, or covalently closed circular. It will be understood that the nucleic acid encodes a functional gene (or part thereof) which enables a glycosyltransferase with the appropriate activity to be produced. Preferably the nucleic acid is in an isolated form; this means that the nucleic acid is at least partly purified from other nucleic acids or proteins.
Preferably the nucleic acid comprises the correct sequences for expression, more preferably for expression in a eukaryotic cell. The nucleic acid may be present on any suitable vehicle, for example, a eukaryotic expression vector such as pcDNA (Invitrogen). The nucleic acid may also be present on other vehicles, whether suitable for eukaryotes or not, such as plasmids, phages and the like.
Preferably the first glycosyltransferase is a an enzyme with a higher affinity for the substrate than said second glycosyltransferase. More preferably said first glycosyltransferase preferentially utilises Type 1 substrates. Still more preferably said first glycosyltransferase is Se (also known as FUT2). Preferably the Se originates or is derived from, or is based on, Se from the same species as the cell in which it is intended to be expressed. Thus, the first glycosyltransferase and the cell in which the enzyme is expressed may each originate from animals of the same species. Such species may be pig, New World monkey, dog or other suitable species. The nucleic acid encoding Se is not necessarily directly derived from the native gene. The nucleic acid sequence for Se may be made by PCR, constructed de novo or cloned.
More preferably Se is of porcine origin or based on the porcine enzyme. This means that the enzyme is based on, homologous with, or similar to native porcine Se.
More preferably the nucleic acid sequence encoding Se is based on, or similar to a 1.3 kb DNA fragment derived from a pig genomic liver. More preferably the nucleic acid sequence encodes the amino acid sequence shown in FIG. 1 (SEQ. ID. NO: 6). Still more preferably the nucleic acid sequence is that shown in FIG. 1 (SEQ. ID. NO: 5).
It is apparent that the Se gene is not expressed in porcine tissues which are of primary interest for transplantation. Thus Se is not expressed in heart, liver, kidney and pancreas, for example. Thus the invention includes the provision of expression of a gene in a tissue where it is not normally expressed, whereby expression results in reduced levels of unwanted carbohydrate epitopes in that tissue and renders an organ composed of that tissue more suitable for transplantation.
The second glycosyltransferase may be any enzyme which produces an unwanted carbohydrate epitope on the cell of interest. This will usually be Gal transferase.
Preferably the cell which expresses the nucleic acid of the invention is a eukaryotic cell. More preferably it is a mammalian cell, still more preferably a New World monkey cell, even more preferably an ungulate cell (pig, sheep, goat, cow, horse, deer, camel, etc.) or a cell from other species such as dogs. Still more preferably the cell is a pig cell.
In a related aspect the invention provides a nucleic acid encoding a first glycosyltransferase which is able to compete with a second glycosyltransferase when said nucleic acid is expressed in a cell which produces said second glycosyltransferase, wherein said first glycosyltransferase is able to utilise more than one substrate, resulting in reduced levels of product from said second glycosyltransferase.
The greater substrate specificity of the first glycosyltransferase means that this enzyme is more efficient at converting substrate to the desired carbohydrate and more effective in reducing the ability of the second glycosyltransferase to produce unwanted carbohydrate epitopes.
Preferably the first glycosyltransferase is Se, still more preferably the Se is as described above.
Still more preferably the first glycosyltransferase has a higher affinity for one or more of its substrates than the second glycosyltransferase.
The invention also extends to isolated proteins produced by the nucleic acid of the invention. It further extends to biologically or functionally active fragments of such proteins.
In another aspect the invention provides a method of producing a nucleic acid encoding a first glycosyltransferase which is able to compete with a second glycosyltransferase for a substrate when said nucleic acid is expressed in a cell which produces said second glycosyltransferase, resulting in reduced levels of product from said second glycosyltgransferase, said method comprising operably linking a nucleic acid sequence encoding a first glycosyltransferase to an appropriate vector or other nucleic acid in order to obtain expression of said first glycosyltransferase.
Those skilled in the art will be aware of the techniques for producing the nucleic acid. Standard techniques such as those described in Sambrook et al may be employed.
Preferably the nucleic acid sequences are the preferred sequences described above.
In another aspect the invention provides a method of reducing the level of a carbohydrate exhibited on the surface of a cell, said method comprising the step of causing a nucleic acid to be expressed in said cell wherein said nucleic acid encodes a first glycosyltransferase which is able to compete for substrate with a second glycosyltransferase and wherein said cell produces said second glycosyltransferase which is capable of producing said carbohydrate.
The cell may be any suitable cell, preferably those described above.
The invention also extends to cells produced by the above method and organs comprising the cells.
The nucleic acid of the invention may be present in the cell with another nucleic acid construct which also down-regulates production of unwanted carbohydrates in the surface of the cells, such as that disclosed in PCT/US95/07554, or that of an International application based on Australian provisional application PO1402 filed Aug. 2, 1996 in the name of The Austin Research Institute.
In another aspect the invention provides a method of producing a cell from one species, such as a donor, which cell is immunologically acceptable to another species which is a recipient, comprising the step of reducing levels of carbohydrate on said cell which cause it to be recognised as non-self by the recipient species, said method comprising causing a nucleic acid to be expressed in said cell, wherein said nucleic acid encodes a first glycosyltransferase which is able to compete for a substrate with a second glycosyltransferase and wherein said cell produces said second glycosyltransferase which is capable of producing said carbohydrate.
The cell may be from any of the species mentioned above. Preferably the cell is from a New World primate or a pig. More preferably the cell is from a pig.
The invention also extends to non-human transgenic animals comprising or harbouring the nucleic acid of the invention.
In another aspect the invention provides an expression unit such as a retroviral packaging cell or retroviral packaging cassette, a retroviral construct or a retroviral producer cell which expresses the nucleic acid of the invention, resulting in a cell which is immunologically acceptable to an animal by having reduced levels of a carbohydrate on its surface, which carbohydrate is recognised as non-self by said animal.
Preferably the animal is a human, ape or Old World monkey.
The retroviral packaging cells or retroviral producer cells may be cells of any animal origin in which it is desired to reduce the level of carbohydrates on the cell surface to make it more immunologically acceptable to a host. Such cells may be derived from mammals such as canine species, rodent or ruminant species and the like.
The invention also extends to a method of producing a retroviral packaging cell or a retroviral producer cell having reduced levels of a carbohydrate on its surface, wherein the carbohydrate is recognised as non-self by an animal, comprising transforming/transfecting the retroviral packaging cell or the retroviral producer cell with the nucleic acid of the invention under conditions such that the chimeric enzyme is produced. The xe2x80x9cchimeric enzymexe2x80x9d means the enzyme encoded by the nucleic acid of the invention.
The term xe2x80x9cnucleic acidxe2x80x9d refers to any nucleic acid comprising natural or synthetic purines and pyrimidines.
The terms xe2x80x9coriginatesxe2x80x9d, xe2x80x9cbased onxe2x80x9d, or xe2x80x9cderived fromxe2x80x9d mean that enzyme is homologous to, or similar to, the enzyme from that species.
The term xe2x80x9cglycosyltransferasexe2x80x9d refers to a polypeptide with an ability to move carbohydrates from one molecule to another.
The term xe2x80x9coperably linkingxe2x80x9d means that the nucleic acid sequences are ligated such that a functional protein is able to be transcribed and translated.
The term xe2x80x9creducing the level of a carbohydratexe2x80x9d refers to lowering, minimising, or in some cases, ablating the amount of carbohydrate displayed on the surface of the cell. Preferably said carbohydrate is in the absence of the first glycosyltransferase of the invention, capable of stimulating recognition of the cell as xe2x80x9cnon-selfxe2x80x9d by the immune system of an animal. The reduction of such a carbohydrate therefore renders the cell, or an organ composed of said cells, more acceptable to the immune system of an animal in a transplant situation or gene therapy situation.
The term xe2x80x9ccausing a nucleic acid to be expressedxe2x80x9d means that the nucleic acid is introduced into the cell (i.e. by transformation/transfection or other suitable means) and contains appropriate signals to allow expression in the cell.
The term xe2x80x9cimmunologically acceptablexe2x80x9d refers to producing a cell, or an organ made up of numbers of the cell, which does not cause the same degree of immunological reaction in the other species as a native cell from the one species. Thus the cell may cause a lessened immunological reaction, only requiring low levels of immunosuppression therapy to maintain such a transplanted organ or no immunosuppression therapy may be necessary.
It is contemplated that the nucleic acid of the invention may be useful in producing the chimeric nucleic acids disclosed in an application based on Australian provisional application PO1402 filed Aug. 2, 1996 in the name of The Austin Research Institute.
The retroviral packaging cell and/or producer cells may be used in applications such as gene therapy. General methods involving use of such cells are described in PCT/US95/07554 and the references discussed therein.